WO2011063595A1 - Depolymerized glycosaminoglycan from thelenota ananas and preparation method thereof - Google Patents

Abstract

Disclosed is a depolymerized glycosaminoglycan from Thelenota ananas(dTHG), weight average molecular weight of which is about 8000 ~ 20000Da, and monosaccharide components of which are acetylgalactosamine(GalNAc), glucuronic acid(GlcUA), fucose(Fuc) or their sulfates(expressed as -OSO₃ ^-), in which molar ratio of GalNAc: GlcUA: Fuc: -OSO₃ ^- is about 1: (1±0.3 ): (1±0.3): (3.5±0.5). Said dTHG is a potent endogenous factor X enzyme inhibitor, which has good anticoagulating and antithrombotic activity, and can be used to prevent and/or treat thrombotic diseases. Also provided is a method for preparing said dTHG, which comprises steps of 1) extracting and obtaining fucosylated glycosaminoglycan(THG) from Thelenota ananas body wall; 2) depolymerizing THG to obtain dTHG by method of peroxide depolymerization or method of peroxide depolymerization catalyzed by catalyst of the fourth period transition metal ions; 3) removing oligomer and/or polymer impurities in dTHG.

Description

 Low poly pineapple gins glycosaminoglycan and preparation method thereof

 The invention belongs to the technical field of medicine, and particularly relates to an oligomeric pineapple gins glycosaminoglycan and a preparation method thereof, a pharmaceutical composition containing the same, and a medicament for preventing thrombotic diseases and/or Use in therapy. The present invention is based on a further invention of the invention patent application entitled "Oli-fucosylated glycosaminoglycan and its preparation method", the application No. 200910110114.0, filed on November 6, 2009 The entire text is incorporated into the specification of the present invention in the form of a reference. technical background

 Fucose-branched Glycosaminoglycan (fiicose-containing glycos-aminoglycan or Fucosylated Glycosaminoglycan, FGAG) or Fucose-branched Chondroitin Sulfate (FCS) refers to a type of extract obtained from the body wall or internal organs of the echinoderms. A special glycosaminoglycan with a sulfated fucose-substituted group (Fan et al., Acta Pharmacea Sinica, 1980, 18(3): 203; Ricardo P. et al., J. Biol. Chem., 1988, 263 (34): 18176; Yutaka K. et al., J. Biol. Chem., 1990, 265:5081).

 Available data show that there is a commonality and difference in the chemical composition of echinoderma-derived FGAG. First, FGAG prepared from different sources and different methods have some common features, namely, FGAG consists of monosaccharides including N-acetylgalactosyl ( GalNAc, A, the N-acetylgalactose chemical name is N-acetyl- 2 deoxy-2-aminogalactose, hereinafter the same), glucuronic acid group (GlcUA, U), fucosyl (Fuc, F) and their sulfates (above Fan Yizeng, 1980; Ricardo P, 1988; and Ken-ichiro Y. et al., Tetrahedron Letters, 1992, 33(34): 4959); wherein GlcUA and GalNAc (or their sulfates) pass β (1-3) and β (1-4) Glycosidic linkages are linked to form a backbone similar to chondroitin sulfate with a [GlcUA β(1-3)- GalNAc β(1-4)-] disaccharide repeating structural unit, while fucose or its sulfate is in the form of a side chain Connected to the main chain.

 FGAGs prepared from different sources and different methods have different degrees of chemical structure differences, such as:

 (1) Differences in the composition ratio of monosaccharides. The monosaccharide composition of FGAG obtained from different species of sea cucumbers, different tissue sources and even different preparation methods may be significantly different. The composition of FGAG monosaccharides from several sources is shown in Table 1.

 Table 1. FGAG monosaccharide composition and sulfate group derived from several sea cucumbers

 Source chemical composition (molar ratio)

 Source of literature

 Sea cucumber species organization A: U: F: -OSO;

^opus Japonicus Body Wall ₁ :! _M : ^: ^ Fan Yizeng, Journal of Pharmaceutical Sciences, _26?

I Ken-ichiro Y, Tetrahedron Letters, 1992, 33:

 1: 0.88 : 0.93 : 4.01

 4959

 1: 1.18 : 2.78: 5.46 Yutaka, J. Biol. Chem., 1990, 265: 5081

1: 0.84 : 2.38: 3.69 ^[b] Yutaka, Biochem. J., 2002, 132: 335

 Viscera 1 : 1.00: 1.00: 4.70 Fan Ji Zeng, Marine Medicine, 1983, (3): 134

 Stichopus variegatus

 Body Wall 1: 1.21 : 1.29 :4.62 Chen Juzhen, Chinese Marine Medicine, 1994, (1 ): 24

 (花剌参)

 Holothuria leucospilota

 1: 0.94 : 0.84 : 3.60 Fan Huazeng, Journal of Pharmaceutical Sciences, 1983, 18 (3): 203

 (玉足海参)

 1: 0.96 : 0.78 : 1.98 Li Haiyan, Chinese herbal medicine, 1999, 22(7): 328

 Holothuria atra

Body wall 1: 1.15 :0.79 : 2.7 [ ^e 】 Tang Xiaoli, Chinese herbal medicine, 1999, 22(5): 223

 (Black Sea Cucumber)

 Holothuria scabr

 Body Wall 1: 1.28 :0.68 : 1.72 , , Food and Fermentation Industry, 2006, 32: 123

 (chain sea cucumber)

Ludwigothurea grisea 1: 1.17 : 2.17: : 2.39 ^[c] Paulo AS, Eur. J. Biochem., 1987, 166:639

Ricardo PV, J. Biol. Chem., 1988, 263: Body wall 1: 0.90:0.97: 2.67 ^[dl

 18176

1: 0.92 : 1.23 : 2.21 Paulo AS, J. Biol. Chem., 1996, 271:23973 The literature is reported in mmol/g (0.81:0.69:1.93:2.99); ^[bl literature in mass percent (%) 3⁄4 ( 16.2 : 20.3: 11.66 : 23.52);

^[c ] The reported ratio of the literature is 0.46: 0.54: 1.00: 1.10; ^{td) The} reported ratio of the literature is 0.33: 0.30: 0.32: 0.88.

 The difference in FGAG composition obtained from different species, tissue sources and different extraction methods is mainly reflected in the large change in the composition ratio of Fuc and sulfate groups. According to the data, it is judged that not only the FGAGs of different species are different, but the difference in extraction methods may also lead to large differences in product composition. For example, YutakaK et al. (1990, 2002) extracted FGAG from the body wall of the sea cucumber with Fan et al. (1980) and Ken-ichiro Y et al. (1992). The former has a higher molar composition of fucose in the FGAG than the latter. 2 to 3 times; FGAG extracted from the same species of sea cucumber. g ea) also has a large change in the proportion of fucose composition (Paulo AS et al., 1987, 1988). The difference in the proportion of fucose composition of FGAG caused by the same source of tissue and different extraction methods/times may be due to the presence of non-GAG fucosan contamination in the FGAG product with a higher proportion of fucose composition, or may be rock In the preparation of FGAG products with low proportion of alginose, the side chain groups are destroyed and lost, and may also be related to the accuracy of the content detection method.

(2) Differences in the structure of the main chain. Like the difference in position and number of sulfate groups in the presence of chondroitin sulfates A, C, D, etc., the position and number of sulfate groups are also present in the main chain structure of FGAG of different species, for example, data display , in the FGAG main chain of the sea cucumber, the 4- and 6-positions of GalNAc have the meaning of succinic acid (Ken-ichiro Y et al., Tetrahedron Letters, 1992, 33: 4959); Jade foot sea cucumber FGAG main chain GalNAc There is only 6-position sulfation and no 4-position sulfation (Fan et al., Pharmacological Journal, 1983, 18 (3): 203); and L. grisea-derived FGAG In the main chain, about 53% of GalNAc is 6-sulfate, a small amount is 4,6-disulfate (about 12%), 4-sulfate (about 4%), and about 31% is not sulfated. GalNAc (Lubor Borsig et al. J. Biol. Chem. 2007, 282: 14984).

 (3) Difference in the degree of side chain fucosyl and sulfation. According to the data, there are three types of side chain fucosyl groups, namely 2,4-disulfate, 3,4-disulfate and 4-sulfate, in 5*. (Hawthorn) and gn a-derived FGAG. Fucosyl; the latter additionally contains about 25% fucose that has not been sulfated, and these fucose bundles are present at the reducing end of FGAG (Ken-ichiro Y. et al., Tetrahedron Letters, 1992) 33: 4959, Paulo AS. et al., J. Biol. Chem., 1996, 271: 23973).

 As shown in Table 1, the degree of sulfation of FGAG obtained from sea cucumber and ginseng is generally higher, while the degree of sulfation of FGAG derived from L. grisea. Black sea cucumber and brown sea cucumber is relatively low.

 Available data show that echinoderma-derived FGAG has a variety of biological activities.

 FGAG from various sources has certain anticoagulant activity (Fan et al., Pharmacological Journal, 1980, 15(5): 263; Zhang Peiwen, Chinese Journal of Pharmacology and Toxicology, 1988, 2(2): 98; Paulo AS. et al., J. Biol. Chem. 1996, 271: 23973); however, these natural FGAGs also have an induction of platelet aggregation activity ( Jia-zeng L. et al, Thromb Haemos, 1988, 54(3): 435; Shan Chunwen, Pharmacology and Clinical Medicine, 1989, 5(3): 33).

 The biological activity of FGAG as reported is also related to the regulation of blood lipids (His-Hisen L. et al., J. Agric. Food Chem., 2002, 50: 3602), anti-arterial injection sclerosis and inhibition of vascular endothelial proliferation (Tapon- Bretaudiere) Et al., Thromb. Haemost, 2000, 84: 332; Masahiko I. et al., Atherosclerosis, 1997, 129: 27; European Patent Application, EP 081 1635), Immunomodulation (Sun Ling et al, Biochemistry and Biophysics) Progress in Studies, 1991, 18 (5): 394; Chen Zuqiong et al., Tianjin Pharmaceutical, 1987,

(5): 278), Anti-tumor {Ji, Chinese Journal of Clinical Oncology, 1992, 19(1): 72; Li Weimin et al, Cancer Clinical, 1985, 12(2): 1 18), and anti-virus (J. Beutler) Etal., Antivir. Chem. Chemother., 1993, 4(3), 167; PCT Patent Application PCT/JP90/00159) and the like.

 The antithrombotic and anticoagulant activities of FGAG and its derivatives derived from sea cucumber and its derivatives, pharmacological action target data show that FGAG has anticoagulant mechanism different from heparin and dermatan dermatan, and its anticoagulation /Antithrombotic targets may involve:

 (1) AT-III: There is an anti-thrombin activity dependent on ΑΤ-ΙΠ (Paulo AS et al., J. Biol. Chem., 1996, 271, 23973; Marcy, Chinese Journal of Hematology, 1990, 1 1 (5): 241); (2) HC-II: the presence of anti-thrombin activity dependent on HC-II (Hideki Nagase et al., Blood, 1995, 85,

(6): 1527; Zhang Guangsen, 1997, 18(3): 127); (3) Ila: inhibition of thrombin (Ila) feedback activator XIII (Nagase H et al., Biochem. J., 1996, 1 19 (1): 63-69);

(4) f.Xase: inhibition of activation of factor X by endogenous factor X enzyme (factor VIII-IX complex) (Hideki Nagase et al., Blood, 1995, 85(6): 1527; JP Sheehan et al. , Blood, 2006, 107(10): 3876 ); ( 5 ) TFPI: including increasing the rate of inhibition of Xa by TFPI, reducing TFPI-Xa Inhibition of TF-VIIa and stimulation of TFPI release ( Hideki Nagase et al., Thromb Haemost, 1997, 78: 864; T. Bretaudiere et al., Thromb Haemost, 2000, 84: 332); (6) Plasmin : Promotes activation of lysogen and promotes thrombolysis (Yang Xiaoguang et al., Chinese Academy of Medical Sciences 4, 1990, 12(3), 187; Yutaka Kariya et al., Biochem.丄, 2002, 132: 335) .

 Although the unique anticoagulant mechanism of sea cucumber FGAG and its good anticoagulant activity make it have important potential application value, FGAG has been difficult to apply in clinical practice so far, mainly because:

 (1) Sea cucumber FGAG has both anticoagulation and activity to induce platelet aggregation. Clinically, anti-coagulation is aimed at antithrombotic, while antiplatelet is another important anti-thrombotic method routinely used clinically. Obviously, for thrombosis, the antithrombotic anticoagulant activity of FGAG and its thrombogenic platelet activation effect are mutually antagonistic, making it difficult to apply to the prevention and treatment of clinical hematological diseases. For example, studies have shown that in an acute diffuse intravascular model of rabbits, the platelet activation effect of FGAG completely counteracts its anticoagulant effect (Li Anguo, Journal of Hunan College of Traditional Chinese Medicine, 1991, 1 1(3): 37) .

 (2) Pharmacological dose of sea cucumber FGAG in vivo intravascular administration can reduce platelet count

(Li Jiazeng et al., ^, 1985, 6(2): 107). Thrombocytopenia, such as heparin-induced immune thrombocytopenia, may trigger a bleeding tendency and may cause severe or even fatal diffuse intravascular coagulation. Available data suggest that thrombocytopenia caused by sea cucumber FGAG may be related to the induction of platelet aggregation and thus the "seizure" of platelets in microvessels (Li Jiazeng et al., Chinese Journal of Traditional Chinese Medicine, 1983, 8(5): 35).

 (3) It is generally believed that a wide range of pharmacological effects are closely related to the bleeding tendency of anticoagulant drugs. Selective targets have become important evaluation indicators for the development of new anticoagulant drugs (KA Bauer, Hematology, 2006, ( 1): 450). Sea cucumber FGAG has different drug targets than other known anticoagulant drugs, but as mentioned above, its target is still relatively broad. The available data have shown that FGAG can produce significant bleeding at anticoagulant dose. Tendency (Paulo AS etal, British Journal of Haematology, 1998, 101: 647).

 Naturally derived sea cucumber FGAG has unique anticoagulant mechanism and potency, on the other hand, it has defects that limit its application. Therefore, it has become one of the important contents of its application research through structural repair to obtain a relatively ideal target product. .

 At present, the chemical structure modification method of FGAG includes peroxidative depolymerization (European Patent Publication EP 040)

8770; Ken-ichiro Y et al., Tetrahedron Letters, 1992, 33: 4959), decalcification or carboxyl reduction (Paulo AS et al., Thrombosis Research, 2001, 102: 167), partial acid hydrolysis (Yutaka Kariya, Biochem. J 2002, 132: 335; Paulo AS et al., Thrombosis Research, 2001, 102: 167) et al. Some progress has been made in these efforts. For example, the study found that the activity of platelet aggregation induced by FGAG of sea cucumber can be reduced with the decrease of molecular weight (Fan et al. Journal of Biochemistry, 1993, 9(2): 146); The anti-thrombin activity of the peroxidative depolymerization product of FGAG may also be reduced (Maxi, Chinese Journal of Hematology, 1990, 1 1 ( 5): 241; Paulo AS et al., J. Biol. Chem. 1996, 271, 23973; Hideki Nagase et al., Blood, 1995, 85 (6): 1527); for source FGAG, molecular weight reduction for its HC-II-dependent anticoagulant live yeast 'I ¹ more significantly affect green (RG Pacheco et al, Blood coagulation and Fibrinolysis, 2000, 11:. 563).

 According to the research data of the FGAG depolymerization product (referred to as DHG), it is still difficult to obtain the anticoagulant activity products of the relative ideal potency and the defect characteristics only by the depolymerization method. For example, data show that the weight average molecular weight of sea cucumber FGAG is as low as about 9000 Da to eliminate its platelet-induced aggregation activity (Fan et al., 1993, 9(2): 146); on the other hand, due to anticoagulant 'f Life is reduced with molecular weight drop (RG Pacheco et al., Blood Coagulation and Fibrinolysis, 2000, 11: 563; Fan et al., J. Biol. Chem., 1993, 9(2): 146). DHG-related pharmacodynamic studies have used DHG with a molecular weight of less than 10,000 Da in the early stage, but in the following ten years of research, the weight average molecular weight of DHG is more than 12,000 ~ 15000 Da ( Hideki Nagase etal., Thromb Haemost , 1997, 77(2): 399; Kazuhisa M et al., Kidney International, 2003, 63: 1548; JP Sheehan et al. Blood, 2006, 107(10): 3876). Obviously, the latter should be necessary to maintain the necessary anticoagulant potency, but for the FGAG, the molecular weight range is clearly safe for eliminating platelet-inducing activity and avoiding thrombocytopenia caused by intravascular administration. Doubt, and this security has not been reflected and verified in relevant research materials.

 Studies have shown that both hydrolyzed side chain fucose and partial desulfation can cause a significant decrease or even disappearance of FGAG anticoagulant activity, and the effect of carboxyl reduction on anticoagulant activity is relatively small, but the bleeding tendency is still significant and affects platelet activity. Unknown (Paulo AS, J. Biol. Chem., 1996, 271, 23973; Paulo AS et al., British J. Haematology, 1998, 101: 647; Paulo AS et al, Thrombosis Research, 2001, 102: 167).

 The research data on the chemical and biological activities of sea cucumber FGAG mainly come from L. grisea and sea cucumber.

FGAG, although there is information on the extraction method of pineapple glycosaminoglycan (Liu Xuexiang, Nanjing University of Traditional Chinese Medicine

 2003, 19(3): 161), but no reports on chemical structure analysis and biological activity have been reported.

Based on the available data, the chemical structure of L. grisea and FGAG can be expressed as follows: The degree of sulfation is different, the type and degree of sulphation of the main chain GalNAc are different, and the types of side chain fucose are basically the same, while the different types are The composition ratio of the chain fucose is different and the degree of acidification of the side chain is different (Ken-ichiro Y et al., Tetrahedron Letters, 1992, 33: 4959; Lubor Borsig et al. J. Biol. Chem. 2007, 282: 14984). According to the anticoagulant titer of two FGAGs relative to heparin and/or low molecular weight heparin, the anticoagulant and antithrombotic potency of sea cucumber FGAG is stronger (Norihiko S et al. Thromb Haemost, 1991, 65(4): 369 Paulo AS et al., British J. Haematology, 1998, 101 : However, none of the above work has clarified the platelet activation effect of the products obtained and the effect of intravenous administration on platelet count, which is the most important factor limiting the application value of sea cucumber FGAG products; secondly, although the relevant data reports these structures The effect of the modified product on certain blood factors (pharmacological targets), but the relationship between structural modification and the pharmacological mechanism of the obtained product is not clear, so it is difficult to judge the extent or extent of structural modification that achieves good pharmacological mechanisms. So far, the effect of sulfation position on the activity and action characteristics of FGAG has not been reported, although it is possible to find a more selective pharmacodynamic effect by changing the type of sulfated structure of the main chain and/or side chain glycosylation. This new type of FGAG is based on the application value. However, under the prior art conditions, although non-selective sulfation or desulfation treatment of glycosaminoglycans is possible, it is difficult to carry out positional selective sulfation or desulfation. Transformation.

 The present invention surprisingly found that the position or type of FGAG side chain fucose thioesterification has an important influence on its biological activity, especially on platelet-induced aggregation activity, through the comparative study on the chemical structure and biological activity of F. chinensis FGAG. As a result, FGAGs of different species origins can have significant differences in application value. In addition, the inventors have now discovered that different degrees of depolymerization and methods have different effects on the intensity of biological effects produced by FGAG through different targets, based on which differences can be obtained with specific target-selective characteristics to obtain treatment for specific diseases. And/or preventive FGAG derivatives.

 The present invention firstly finds that Fucosylated glycos-aminoglycan from Thelenota ananas (THG) has special chemical structural features, and its side chain fucose type is different from FGAG which has partially or partially clarified chemical structure. For example, JL grisea and sea cucumber FGAG; THG is also unique in its biological activity, and its platelet activation activity is much lower than that of FGAG derived from sea cucumber and sea cucumber.

 The present invention confirms that THG has the activity of inhibiting endogenous factor X enzyme (f.Xase) and HC-II-dependent antithrombin (f.IIa), and firstly elucidates the degree of depolymerization of THG and the pharmacological action. On the basis of the correlation of the activity intensity, the present invention obtains a THG depolymerization product (Depolymerized THQ dTHG) having a high ratio of inhibition of f.Xase/Ila activity (potency ratio). That is to say, the present invention uses a specific chemical and biological activity of THG as a starting material, based on the correlation law between depolymerization and biological activity, to prepare a good anticoagulant titer and has a special defect. A selectively characterized dTHG product, the dTHG has no activity to induce platelet aggregation, and no platelet count is reduced in the case of high doses and repeated administration. Summary of the invention

It is an object of the present invention to provide an oligomeric pineapple glycosaminoglycan having a specific chemical structure and good anticoagulant potency, which does not have an activity of inducing platelet aggregation and does not cause a decrease in the number of platelets. Another object of the present invention is to provide a process for preparing the above oligomeric pineapple glycosaminoglycan.

 Still another object of the present invention is to provide a pharmaceutical composition comprising the above oligomeric pineapple glycosaminoglycan and its use in the preparation of a medicament for preventing and/or treating a thrombotic disease.

 The present inventors have found that the Glycosaminoglycan from Thelenota ananas (THG) derived from the pineapple ginseng has special features such as the known FGAG, such as the composition of monosaccharides including GalNAc, GlcUA and Fuc, etc. Its chemical structural features, its side chain fucose type, and degree of sulfation are different from the sea cucumber FGAG that has been elucidated or partially elucidated. For example, grisea and sea cucumber FGAG mainly include three structural types of side chain fucose, namely fucose-2,4-disulfate group, -3,4-disulfate and -4-sulfate group. THG mainly includes fucose-2,4-disulfate group, -3-sulfate group and -4-sulfate group. Consistent with this, the present invention confirmed that THG has an activity of inhibiting endogenous factor X (f.Xase) and HC-II-dependent antithrombin (f.IIa), and found that THG is also biologically active. It exhibits specificity, for example, its platelet activation activity is much lower than that of FGAG derived from sea cucumber and sea cucumber.

 In addition, the inventors have surprisingly found that the position and type of FGAG side chain fucose sulfation has an important influence on its biological activity, especially on platelet-induced aggregation activity, by comparing the chemical structure and biological activity of THG.

 Based on the above research, the present invention uses THG with special chemical structure and biological activity as a starting material, based on the correlation law between depolymerization and biological activity, and has a good anticoagulant titer and a special target. Point-selective feature of oligomeric pineapple glycosaminoglycan (Depolymerized THG, dTHG).

 According to one aspect of the invention, the oligomeric pineapple glycosaminoglycan (dTHG) is a depolymerization product of fucosylated glycosaminoglycan (THG) derived from Thelenota ananas, which has the formula Structure:

In the formula (I): -OR is a hydroxyl group (-OH), a sulfate group (-OS0 ₃ - ), or a sulfated fucosyl group as shown by the formula (Π):

In the formula (Π): -OR defines the same formula (1).

Wherein: the monosaccharide composition includes N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), fucose (Fuc), or its sulfate (expressed as -OSC), in molar ratio, GalNAc: GlcUA: The ratio of Fuc: - OS0 ₃ is approximately 1: (1 ± 0.3): (1 ± 0.3): (3·5 ± 0.5), and the weight average molecular weight (Mw) of dTHG is about 8000 ~ 20000 Da. A preferred molecular weight range is from 10,000 to 18,000 Da, more preferably from 12,000 to 16000 Da.

 Studies have shown that the dTHG of the present invention has a higher anti-f.Xase/anti-f.IIa activity intensity ratio (potency ratio), a higher anti-Xase/prolonged APTT titer ratio, and no activity for inducing platelet aggregation, and High doses and repeated administrations do not occur with a reduction in platelet count and can be used to prevent and/or treat thrombotic diseases.

 According to another aspect of the present invention, there is provided a method of preparing the above oligomeric pineapple glycosaminoglycan, which comprises the following steps:

 1) Extraction: obtaining fucosylated glycosaminoglycan (THG) from the body surface of pineapple;

 2) depolymerization: depolymerization step 1) obtained THG to obtain oligofucosylated glycosaminoglycan (dTHG);

3) Refining: The dTHG of the desired molecular weight is collected and purified to remove low molecular and/or high molecular impurity components therein.

 Specifically, step 1) may generally include steps of cutting/pulverization, enzymatic hydrolysis, alkaline hydrolysis, decolorization, separation, and the like. Pineapple can be fresh or dried to the internal organs. In order to achieve the purpose of increasing the extraction yield, the dried pineapple ginseng product generally needs to be cut into a sheet or a small piece, and then immersed in water. For the fresh product, it can be cut and directly pulverized into a suspension, and then Enzymatic and/or alkaline treatment is carried out.

 In the enzymatic hydrolysis step, broad-spectrum proteases are generally selected, including animal-derived pepsin, trypsin, complexase or crude enzyme preparations, plant and/or microbial-derived proteases such as papain, actinase and the like. Enzymatic conditions such as temperature, time, pH and amount of enzyme are determined according to the nature of the protease used.

 In the alkaline hydrolysis step, a strong base such as potassium hydroxide or sodium hydroxide may be selected, and the amount is generally selected so that the alkali concentration in the extract reaches about 0.5 to 2 N (equivalent concentration), and the reaction temperature is generally selected from room temperature to 70 ° C. The alkali treatment time may be about 0.5 to 3 h.

After the enzymatic and/or alkali-treated extract is subjected to inactivation and/or neutralization treatment, the insoluble matter is removed by centrifugation and/or filtration, and the resulting supernatant may be used or combined with a lower alcohol/ketone such as ethanol or acetone. , or a potassium salt such as an acetic acid clock, or an acid mucopolysaccharide precipitant such as a glycosaminoglycan in a cetylpyridinium extract extract, The resulting precipitate can be dried to a crude extract or directly to the next step without drying. The crude glycosaminoglycan extract may be optionally subjected to decolorization, fractional precipitation, gel chromatography and/or ion exchange chromatography. The invention preferably uses decolorization of hydrogen peroxide combined with ethanol-potassium acetate fractionation precipitation method (see Fan et al., Journal of Pharmaceutical Sciences)

1983, 18 (3): 203) Obtain relatively pure pineapple ginseng glycosaminoglycan, THG.

In the present invention, the fucosylated glycosaminoglycan (THG) derived from the lentil (Thelenota a leg as) has the common characteristics of some known fucosylated glycosaminoglycans, for example, the constituent monosaccharides including acetamidogalactose (GalNAc), glucuronic acid (GlcUA), fucose (Fuc), etc., but the degree of sulfation (expressed as -oso ₃ -) is different from the known fucosylated glycosaminoglycans. In the integer (or half) molar ratio, the composition ratio of the composition monosaccharide and sulfate group of THG is GalNAc: GlcUA: Fuc: -OS0 ₃ - close to 1: 1 : 1 : 3.5, further, about 1: (1) ±0.3 ): ( 1 ± 0.3 ): ( 3.5 ± 0.5 ).

 In step 2), the depolymerization method of THG may be selected from a hydrogen peroxide depolymerization method or a peroxide depolymerization method catalyzed by a transition metal ion in the fourth period. The latter is preferably used in the present invention, which is disclosed in the patent application filed on November 6, 2009 by the applicant, entitled "Oltra-fucosylated glycosaminoglycan and its preparation method", application No. 200910110114.0 Detailed description, it is to use a catalyst containing a fourth period transition metal ion in an aqueous medium to catalyze the depolymerization of the peroxide to obtain an oligomeric pineapple ginseng fucose glycosaminoglycan. Specifically, the method includes the following steps:

 2.1) In the presence of a catalyst for transition metal ions in the fourth cycle, in the aqueous phase shield, a peroxide is added to depolymerize the fucose-derived glycosaminoglycan (THG) derived from sea cucumber;

 2.2) Stop the reaction and collect the oligomeric fucosylated glycosaminoglycan (dTHG) of the desired molecular weight range. Wherein, the depolymerization reaction is carried out in an aqueous medium, and the peroxide can generate a free radical in the reaction system, and the THG is cleaved by a free radical chain reaction to form a dTHG product. The peroxides include, but are not limited to, peroxyacetic acid, hydrogen peroxide, 3-chloro-perbenzoic acid, cumene hydroperoxide, sodium persulfate, benzoyl peroxide, and salts or esters thereof, preferably Hydrogen peroxide.

 The shield fraction of the THG in the reaction system is about 0.05%-15%, and the shield fraction of the peroxide in the reaction system is about 0.5% to about 30%. During the depolymerization reaction of FGAG, the peroxide reactant may be added to the reaction system at one time before the reaction, or the peroxide reactant may be gradually added to the reaction system in a continuous or intermittent manner. The present invention preferably continuously adds the peroxide reactant to the reaction system in a controlled rate.

The metal ion as a catalyst is a fourth periodic transition metal ion, including Cu+, Cu ²⁺ , Fe ²⁺ , Fe ³⁺ , Cr ³⁺ , Cr ₂ 0 ₇ ² \ Mn ²⁺ , Zn ²⁺ , Ni ^{2 . +,} etc., these metal ions may be used singly or in combination with each other as a composite catalyst. Among them, preferred catalysts are Cu+, Cu ²⁺ , Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , and most preferably Cu ²⁺ . Since metal ions are not independent chemical agents, inorganic or organic salts of these metal ions are actually used. In the reaction system, the concentration of the metal ion may range from about 1 nmol/L to 0.1 mol/L, and the preferred concentration range is from 10 μιηοΙ/L to 10 mmol/L.

The conventional process parameters of the depolymerization reaction process are: temperature range of 10 ° C ~ 75 ° C; The reaction time is from 20 minutes to 8 hours; the reaction can be carried out under normal pressure or under pressure; it can be carried out under the protection of nitrogen or inert gas, or in the atmosphere under normal pressure.

 At the end of the reaction, a chelating agent is optionally added to the reaction system to chelate it with the metal ion catalyst to suppress the catalytic reaction rate, and then the reaction is terminated by a technique such as cooling or precipitation of an organic solvent. A chelating agent refers to a physical shield capable of forming a chelate with a metal ion, including but not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 3-propylenediaminetetraacetic acid (PDTA). , triacetinylamine (NTA) or a salt thereof. The process of the invention is preferably disodium edetate or a hydrate thereof. The reaction product precipitation method is a method of adding a organic solvent to precipitate a polysaccharide substance from a reaction system directly or under the condition of further adding an inorganic salt such as an acetic acid clock, and the organic solvent includes a low carbon such as decyl alcohol, ethanol or acetone. Alcohol/ketone, of which ethanol and acetone are preferred.

 The above method can significantly improve the reaction conditions of the peroxide depolymerization fucosylated glycosaminoglycan, that is, when the same peroxide reactant and the prototype FGAG starting material are used to prepare dFG of the same or approximately equal molecular weight, the reaction conditions are similar at temperature and the like. In the case of the direct peroxide depolymerization method (i.e., the peroxide depolymerization method in the absence of a metal ion catalyst), the method can increase the reaction rate and shorten the reaction time course; similarly, the conditions for controlling the reaction time Next, the method can significantly lower the temperature required for the reaction, so that the desired reaction can be completed at room temperature.

 In the repeated preparation of dTHG, the batch-to-batch difference of the product obtained by the method is significantly reduced compared with the direct oxide depolymerization method under the condition that the reaction conditions are the same or similar, and the molecular weight of the obtained oligomerization product and the target molecular weight are A difference of less than 5% can significantly improve the repeatability and controllability of dTHG preparation, and the oligomerization product has better quality uniformity. This may be related to a decrease in activation energy caused by the catalyst and a relatively stable reaction rate, and may also be related to a decrease in the reaction temperature or a shortened reaction time course, and an improvement in the controllability and stability of the reaction conditions and the reaction conditions.

 Further, in order to further improve and improve the controllability of the depolymerization reaction, a certain concentration of inorganic and/or organic salts may be selectively added to the metal ion-catalyzed peroxide depolymerization reaction system. The inorganic and/or organic salts include salts of metal elements (such as alkali metals, alkaline earth metal elements, etc.) with halogens, organic acids, and the like, salts of organic or inorganic acids with organic bases, and complex salts thereof in combination with each other. Among them, sodium chloride, potassium chloride, sodium acetate, sodium acetate trihydrate, and potassium acetate are preferred. In the present invention, a preferred salt concentration of the inorganic and/or organic salt for improving the depolymerization reaction rate and reaction controllability is from about 0.1 mmol/L to about 1.0 mol/L.

 In the present invention, the collected dTHG has a weight average molecular weight (Mw) of about 8,000 to 20,000 Da, and a preferred molecular weight range of 10,000 to 18,000 Da, more preferably 12,000 to 16,000 Da.

In step 3), the collected dTHG can be purified by methods known in the art to remove low and/or high molecular impurity components. Purification methods include, but are not limited to, removal of low and/or high molecular impurity components by dialysis, preparation of THG salts by ion exchange, and/or purification by gel chromatography/anion exchange chromatography. Further, the method for preparing an oligomeric pineapple glycosaminoglycan of the present invention may comprise the steps of:

 4) DTHG obtained in the drying step 3). The drying method may be selected from vacuum drying or freeze drying, and the present invention is preferably dried by freeze drying.

 The monosaccharide composition and the sulfate group composition ratio of the dTHG prepared by the above method of the present invention can be detected by chemical colorimetry, infrared spectroscopy (IR) method and nuclear magnetic resonance spectroscopy (NMR) method known in the art ( Zhang Weijie, Biochemical Research Technology of Sugar Complex (Second Edition), Zhejiang: Zhejiang University Press, 1999; Applicant's aforementioned invention patent application CN200910110114.0).

 Since the dTHG of the present invention has a sulfate group and a carboxyl group, it may form a pharmaceutically acceptable salt or ester with an inorganic ion or an organic basic group, wherein the pharmaceutically acceptable inorganic salt of the dTHG may be an alkali metal and / or alkaline earth metal salt, preferably a sodium salt, a potassium salt or a calcium salt. It will be apparent that such salts or esters are also included within the scope of the invention.

 According to still another aspect of the present invention, there is provided a pharmaceutical composition comprising dTHG of the present invention or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable excipient. The pharmaceutical composition can be prepared into various dosage forms such as oral preparations (including solid and liquid preparations), injections (including injections and lyophilized powder injections), but the inventors have found that oral absorption of dTHG is weak, and subcutaneous injection or the like. Administration by injection route has good bioavailability. Therefore, the pharmaceutical composition of the present invention is preferably prepared as an injection, and since dTHG has good water solubility, its solution type preparation and lyophilized preparation can be easily prepared by a technical method commonly used in the art.

 The dTHG of the present invention is a potent endogenous factor X enzyme inhibitor having good anticoagulant and antithrombotic activity, and thus the above pharmaceutical composition containing dTHG can be used for preventing and/or treating thrombotic diseases, for example, various techniques. Post-anticoagulant therapy, prevention and treatment of various arteriovenous thrombosis, prevention and treatment of thrombotic-related cardiovascular and cerebrovascular diseases. The use and dosage of the prevention/and/or treatment of different diseases should be determined by the clinician. DRAWINGS

 Figure 1 : NMR spectrum of the THG and dTHG samples, in which the water peaks in the dTHG-6 and dTHG-9 spectra are suppressed;

Figure 2: shows the 'H- ¹ !! COSY spectrum of dTHG (A) and oligosporin FGAG (ie dSJG, B); the signal indicated by the arrow shows the side chain fucose structure of two oligomeric FGAG products Type difference: Figure A shows the fucose-3-sulfate-related signal contained in dTHG, Figure B shows the fucose-3,4-disulfate-related signal contained in dSJG; the signal in the box is fucose The hydrogen signal associated with -3-sulfate and fucose-3,4-disulfate; the signal within the bismuth is the 4-position hydrogen signal of fucose-3,4-disulfate.

Figure 3: shows the ¹³ C NMR and DEPT spectra of THG and sea cucumber FGAG (ie SJG ); Figure 4: shows the NMR homonuclear/heteronuclear correlation spectrum of dTHG-6;

Figure 5: shows that different molecular weights of dTHG inhibit f.Xase activity, relying on HC-II anti-Ila activity And a potency relationship for prolonging the APTT time, wherein the box defining portion is the molecular weight range of the dTHG of the present invention;

 Figure 6: The effect of dTHG and low molecular weight heparin sodium (LMWH) on bleeding time in experimental animals. detailed description

 The invention is further understood by the following detailed description of the embodiments of the invention,

Example 1 THG Extraction, Depolymerization and Purification

1.1 Materials

 Pineapple ginseng, also known as the ginseng (Theienota ananas, commercially available products, to the visceral dry body wall. Stichopus japonicus, commercially available products, to the visceral dry body wall.

 Jade sea cucumber H. leucospilota, a commercial product, goes to the visceral dry body wall.

Papain: 8xl0 ⁵ U / g, Guangxi Nanning Pangbo Biological Engineering Co., Ltd.

 Cation exchange resin: 001x7 strong acid styrene cation exchange resin, Tianjin Nankai University Resin Factory.

Reagents such as KOH, KCOCH ₃ , H ₂ 0 ₂ , and ethanol are commercially available analytical reagents.

1.2 extraction

Take 20 kg of dried pineapple ginseng, cut into thin slices with a thickness of about 1.5 mm, place them in a sandwich reaction tank (300 L), and add 200 L of water to stir. Solid sodium hydroxide was added to a concentration of 0.5 M with stirring, and alkalized at 60 ° C for 2 h. After cooling, 6N hydrochloric acid was adjusted to pH 6-7, 100 g of papain was added, and the reaction was stirred at 50 ° C for 6 hours, and the temperature was raised to 100 ° C for 10 min. After cooling, the pH was adjusted to 2 (precipitated protein) with 6 N hydrochloric acid, and allowed to stand at 2 to 8 ° C for about 4 h, and centrifuged to precipitate. The obtained supernatant was adjusted to a pH of about 7, and ethanol was added to a final concentration of 70%. The mixture was allowed to stand, centrifuged, centrifuged and dissolved in about 30 times (v/v) water, and centrifuged to remove insoluble matter to 2 M NaOH. The pH was adjusted to about 10, H ₂ 0 _{2 was added} to a final concentration of about 3 % (v/v), and the reaction was stirred at 50 ° C for 2 h (decolorization). The reaction solution was added with potassium acetate to a final concentration of 0.5 mol/L, and ethanol was added to a final concentration of 30%. The mixture was allowed to stand, centrifuged, centrifuged and dissolved in 20 times (v/w) water, and centrifuged to remove insoluble matter. Add acetic acid clock to a final concentration of 2.5 mol / L, stand, centrifuge, centrifuge to precipitate ethanol for 2 times, decompress the residual ethanol, water-soluble freeze-drying, that is, the pineapple glycosaminoglycan (THG) about 162 g.

 As a reference, 2 kg of sea cucumber and jade foot sea cucumber were extracted with the above conditions, and the sea cucumber FGAG (SJG) 20.2 g and the jade foot sea cucumber FGAG (HLG) 15.6 g were obtained respectively.

1.3 Depolymerization and refining

50 g of THG prepared by the above method was added, and 1825 ml of water containing 122 g of sodium acetate trihydrate and 60 g of sodium chloride was added, and 120 ml of a copper acetate solution having a concentration of 0.0668 mol/L was added thereto, and the mixture was stirred and mixed. 35 Under stirring ° C water bath, in about 2 hours time course was added dropwise to 126ml / h rate of 10% (v / v) of H ₂ 0 _2. The pH of the reaction was controlled throughout the process from 7.2 to 7.8. The reaction was continuously stirred under the above conditions for about 5 hours, and the reaction was carried out at 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 hours after the start of the reaction. The liquid is about 180 ml.

Each of the fractions to be separated was added with 0.5 g of Na ₂ EDTA immediately after the removal, and the pH was adjusted to 6.5 to 7.0 with 15% acetic acid, and then 2.5 times by volume (about 450 ml) of ethanol was added to the reaction solution, and allowed to stand. The precipitate was obtained by centrifugation. The precipitate was dissolved in 100 ml of water, re-alcoholized (250 ml of 95% ethanol), and the precipitate obtained by centrifugation was washed twice with 50 ml of ethanol, and the ethanol was evaporated under reduced pressure to dissolve in about 30 times (v/w) water, 0.45 μm film. After suction filtration, the filtrate was passed through a Na ⁺ type cation exchange resin column (040 mm X 250 mm ), and the effluent was collected, dialyzed against a dialysis membrane with a molecular weight of 3500 Da for 6 hours, and the dialysis retention solution was freeze-dried to obtain a solution corresponding to each depolymerization time point. The polydip products dTHG-1 ~ dTHG-10, the corresponding yield at each time point is in the range of about 3.0 ~ 3.5 g, and the total amount of the product is 33.2 g.

 As a reference, lOg SJG and 10g HLG were respectively taken and operated under the above depolymerization conditions, and the time point for depolymerizing the reaction liquid was set to two, that is, 3 hours and 5 hours after the start of the reaction. The obtained depolymerized products were recorded as dSJG-1, dSJG-2 and dHLG-1, dHLG-2, and the total amount of dSJG was 7.5 g, and the total amount of dHLG was 8.0 g.

 1.3 Physicochemical properties of dTHG, dSJG and dHLG were tested and compared

 Sample: THG, dTHG-1 ~ dTHG- 10

 Reference samples: SJG, dSJG-1 ~ dSJG-3; HLG, dHLG-1 ~ dHLG-4

 Molecular weight detection: HPGPC-LALLS method

Optical rotation detection: Chinese Pharmacopoeia (2005 edition) two appendix VIE method. WZZ-1S automatic polarimeter, sodium source (λ ₅₈₉ . _3ηπι ), sample tube ldm.

 Intrinsic viscosity test: Chinese Pharmacopoeia (2005 edition) two appendix VI G third method.

 Monosaccharide composition test: acetylgalactose (GalNAc): Elson-Morgon method (Zhang Weijie, Sugar Complex Biochemical Research Technology (Second Edition), Zhejiang: Zhejiang University Press, 1999, 19-20); Glucuronic acid ( GlcUA ): carbazole method;

 Fucose (Fuc): Calculate the GalNAc/Fuc molar ratio according to the NMR 曱 base peak integrated area as described in Example 2;

Sulfate (-OSCV): Determination of Carboxylate/Sulfuric Acid Molar Ratio by Conductivity Method (Zhang Weijie, Biochemical Research Technology of Sugar Complex (Second Edition), Zhejiang: Zhejiang University Press, 1999, 409-410), as GlcUA / -OS0 ₃ - Composition ratio.

 Results: See Table 2.

 Table 2. Chemical composition and physical and chemical properties of various samples

 Molecular weight optical rotation characteristic viscosity monosaccharide composition (molar ratio)

^†O ° (MW) ([a] _D ²⁰ , C=l%) ( O.lM NaCl, ml/g ) GalNAc: GlcUA: Fuc: -OS0 ₃ " THG 65820 -59.1° 46.7 1.00 1.16 1.01 3.60 dTHG-1 46070 -58.3° 29.3 1.00 1.09 1.00 3.52 dTHG-2 33260 -58.2° 19.2 1.00 1.12 1.02 3.61 dTHG-3 25380 -59.5° 13.5 1.00 1.03 0.98 3.45 dTHG-4 19650 -57.3° 9.64 1.00 1.06 1.01 3.50 dTHG-5 17150 -55.7° 8.07 1.00 1.15 0.97 3.42 dTHG-6 13950 -53.7° 6.17 1.00 1.03 0.96 3.53 dTHG-7 1 1580 -58.4° 4.84 1.00 1.08 1.00 3.48 dTHG-8 10260 - 55.9° 4.12 1.00 1.04 0.99 3.56 dTHG-9 8549 -59.0° 3.25 1.00 1.06 0.97 3.55 dTHG-10 6725 -56.9° 2.37 1.00 1.01 0.95 3.43

 SJG 68740 -64.2° 54.6 1.00 1.05 1.02 4.12 dSJG-1 14930 -63.5° 16.9 1.00 0.98 0.95 4.09 dSJG-2 9300 -60.3° 8.5 1.00 0.95 0.93 3.93

HLG 51500 -48.9° 36.5 1.00 0.97 0.89 2.01 dHLG-1 13320 -47.3° 5.70 1.00 0.97 0.85 1.98 dHLG-2 9790 -47.6° 4.24 1.00 0.95 0.84 1.96 From the results in Table 2, dTHG, dSJG, dHLG and its prototype polysaccharide The molar ratios of GalNAc: GlcUA: Fuc in THG, SJG and HLG are close to 1: 1 : 1 , while the content of sulfate base is different, among which the content of SJG/dSJG is higher and the content of HLG/dHLG is lower; There are also large differences in the optical rotation range and intrinsic viscosity of the FGAG from which the source is derived.

For THG/dTHG, the monosaccharide composition ratio varies little in the products prepared from the same batch of sea cucumber raw materials, while the THG and/or dTHG products prepared from different batches of raw materials vary in the range of monosaccharide ratios. Broadening, in general, its GalNAc: GlcUA: Fuc: -OS0 ₃ - molar ratio is usually still in the range of 1: (1 ± 0.3): (1 ± 0.3): (3.5 ± 0.5).

The above results also show that the ratio of fucose to sulfate content in the product obtained by the transition metal ion-catalyzed peroxide depolymerization method is small, and the reducing end of the product is known by iH- ¹ !! COSY detection and analysis. Mainly GalNAc, which is basically consistent with the results of the applicant's aforementioned invention patent application CN200910110114.0.

Example 2 THG/dTHG Spectral Analysis

 Test samples: THG, dTHG-6, dTHG-9, source as in Example 1.

 Reference sample: SJG, SJG-1, source as in Example 1.

Detection spectrum: NMR; ¹ H- ¹ H COSY; Ή-Ή TOCSY; ^l H- ^l H NOESY; ^l3 C-NMR; DEPT-135°; 'H- ¹³ C HSQC; - ¹³ (HMBC.

Test conditions: solvent, D ₂ 0 , 99.9 Atom% D (Norell); internal standard, trimethylsilyl-propionic acid (TSP-d4); temperature, 45 °C.

Instrument, AVANCE AV 400 superconducting nuclear magnetic resonance spectrometer (400 MHz, Bruker, Switzerland). Spectrum: See Figure 1 to Figure 4. Result analysis:

 (1) Comparison of the spectra of THG and dTHG:

 Figure 1 shows the 'Η NMR spectrum of THG, dTHG-6, and dTHG-9, in which the water peaks in the dTHG-6 and dTHG-9 spectra were suppressed. It can be seen from Fig. 1 that the signal characteristics of THG, dTHG-6 and dTHG-9 are basically the same. When the molecular weight is low, the signal is clearer. It can be seen that the NMR signal characteristics before and after THG depolymerization remain stable, indicating that the degree of polymerization is reduced. The basic chemical structure before and after THG depolymerization did not change significantly.

 (2) Comparison of THG and SJG spectra:

Referring to Figures 2 to 4, Figure 2 shows the COSY spectrum of dTHG (A) and dSJG (B); the signal indicated by the arrow shows the difference in the structure of the fucose structure of the two oligomeric FGAG products: A Figure is dTHG Contains the fucose-3-sulfate-related signal, B is the fucose-3,4-dikecoate-related signal contained in dSJG; the signals in the box are fucose cyanate and fucoal The hydrogen signal of the sugar-3,4-diacid ester; the signal within the double circle is the 4-position hydrogen signal of fucose-3,4-disulfate. Figure 3 shows the ¹³ C NMR and DEPT spectra of THG and SJG. Figure 4 shows the NMR homonuclear/heteronuclear correlation spectrum of dTHG-6.

 According to the comprehensive graphic data, the main chain structure of THG and SJG is basically similar. The significant difference between the two is: As shown in Figure 2, the strong signal of dSJG 3,4-disulfate fucose is very weak in the THG spectrum. Or absent; the latter signal belonging to 3-sulfate fucose does not appear in the SJG spectrum, indicating that there is a significant difference in the type of side chain substituents.

 Referring to Figure 3, the carbon signal of the 6-position with and without sulfate-substituted GalNAc appears at about 70 ppm and about 64 ppm, respectively, since the 6-position carbon of GalNAc belongs to the secondary carbon, and its signal peak in the DEPT135° spectrum. It is a peak. It can be seen from Fig. 3 that there is still a small amount (less than about 10%) of the GalNAc in which the 6-position hydroxyl group is not substituted by the sulfate group in the THG main chain, and there is substantially no 6-sulfate-free-substituted GalNAc in the SJG main chain. This further demonstrates the difference in the type of side chain substituents between THG and SJG.

Furthermore, it is known from the literature that the THG of the present invention differs from other known side chain substituents of sea cucumber FGAG, for example, data (Paulo AS et al., J. Biol. Chem. 1996, 271: 23973; Lubor Borsig etal, J. Biol Chem 2007 , 282, 14984) ^I3 C NMR spectrum and the following display, FGAG L. grisea derived from about 35 percent GalNAc 6-position sulfate group is not substituted;... information ( Fan et al., Pharmaceutics Journal, 1983, 18 (3): 203) showed that the GGANA main chain of G. japonicus GGANA had only 6-position sulfated and no 4-sulfated.

In summary, the THG of the present invention has a chemical structure of FGAG derived from other species of known structure, which first manifests in the difference in the structure type of the side chain fucose substituent, that is, mainly containing fucose- 2,4-disulfate group, -3-sulfate and -4-sulfate group, containing no or less fucose-3,4-disulfate; its main chain structure and other species FGAG also has varying degrees of difference. (3) dTHG-6NMR spectrum data attribution: The spectrum attribution is shown in Table 3, and several related spectra are shown in Figure 4.

Table 3. THG-6 'H/^C-NMR spectrum data and attribution

 In the above table: GalNAc4S6S represents 4,6-disulfate-N-acetylaminogalactosyl; GalNAc4S represents 4-acid-N-acetylaminogalactosyl; Fuc-2S4S represents 2,4-disulfate fucose Fuc-3S represents 3-sulfate fucose group; Fuc-4S represents 4-sulfate fucose group; Fuc-OS represents fucosyl group without acid group. Brackets [] indicate that the data in it is difficult to confirm due to excessive overlap of signals.

Example 3 Detection of THG/dTHG biological activity

3.1 Platelet-induced activity assay

 Sample: dTHG-1 ~dTHG-10, source as in Example 1

 Reference sample: dSJG-1 ~ dSJG-4, source same as Example 1

METHODS: New Zealand white rabbits were bled with abdominal aorta, anticoagulated with 3.8% sodium citrate (1:9), platelet-rich plasma (PRP) was obtained by centrifugation at 200 xg for 8 min, and platelet-poor plasma was obtained by centrifugation at 1500 x g for 10 min. The PRP platelet count is approximately 4.0 X 10 ⁵ /mm ³ . The Bron method (Bom GVR. Nature, 1962, 194: 927) platelet accumulator measures the effect of samples on platelet aggregation. The test was performed with saline as a blank control. The final concentration of the sample was 200 g/ml, and the test was repeated three times to calculate the mean platelet aggregation rate.

 Results: See Table 4.

Table 4. Comparison of different FGAG-induced platelet aggregation activities NS THG dTHG-1 dTHG-2 dTHG-3 dTHG-4 Platelet aggregation rate 2.7 ± 4.1 28.1 ± 7.8 16.3 ± 5.6 12.8 ± 4.9 9.3 ± 4.8 3.9 ± 2.8 Sample dTHG-5 dTHG-6 dTHG-7 dTHG-8 dTHG-9 dTHG-10 Platelet aggregation rate 1.9 ±2.9 1.1 Soil 1.3 2.9 ±3.3 2.4 ±3.3 1.2 ±2.5 3.4 ±4.3 Sample SJG dSJG-1 dSJG-2 SJG dHLG-1 dHLG- 2 Platelet aggregation rate 58.4 ± 13.6 22.7 ± 8.5 5.3 ± 5.5 53.6 ± 17.4 18.3 ± 8.6 4.6 ± 5.4 By comparing the platelet-inducing activity of FGAG derived from the above-mentioned pineapple, sea cucumber, jade foot sea cucumber and its different degree of depolymerization products, It can be found that for the prototype FGAG, the activity of THG-induced platelet aggregation is much lower than that of SJG and HLG, indicating that the FGAG structure differences of different species are Platelet activity has a significant effect. In view of the structural differences between THG and SJG and HLG, mainly in the type of side chain acidified fucosyl group, it is speculated that the difference in platelet activity is due to the difference in fucose side chain, THG side chain fucoid The glycosyl type/feature effectively weakens its platelet aggregation activity. In terms of molecular weight, the platelet-inducing activity of dTHG has disappeared when the weight average molecular weight of THG is as low as about 20,000 Da, and the molecular weight of SJG and HLG needs to be low at about 9,000 - 12,000 Da to avoid platelets at high concentrations. Activation effect, this and the literature (Fan et al.,

Zhi, 1993, 9(2): 146) The results are basically the same, which indicates that dTHG has better application value than prototype THG.

 3.2 anticoagulant activity in vitro detection

 Sample: dTHG-1 ~ dTHG-10, source as in Example 1

 Reference sample: dSJG-l ~ dSJG-4, source same as example 1

 Low molecular weight heparin sodium (LMWH): 3500 - 5500 Da, 0.4 mix 4000AxaIU, Anofi - Aventis (France).

 Reagents: Thrombin (Ila): 123 NIH U/mg, sigma (USA).

 Thrombin assay chromogenic substrate (S): 25 mg/vial, HYPHEN BioMed (France).

 Heparin Cofactor II (HC-II): 100 μΐ'Άΐ, HYPHEN BioMed (France).

 Factor VIII (f.VIII): 200 IU/branch, Green Cross (China) Biological Products Co., Ltd. f.VIII test kit: reagents include Reagents: R1: Human Factor X; R2: Activeation Reagent, human Factor IXa, containing human thrombin, calcium and synthetic phospholipids; R3: SXa-11, Chomogenic substrate, specific for Factor Xa; R4 :

Tris-BSA Buffer; HYPHEN BioMed (France).

 Rabbit platelet-poor plasma, Guangzhou Rui Te Biotechnology Co., Ltd.

 APTT assay kit (ellagic acid), Shanghai Sun Biotechnology Co., Ltd.

 Instruments: Microplate reader, Bio-Rad 680 (USA); BICO-dual channel blood coagulation instrument, Minivolt (Italy).

 Method:

(1) Inhibition of endogenous factor X enzyme (f.Xase, Tenase) activity detection: 检测 The detection method established by the f.VIII detection kit combined with the f.VIII reagent. Serial concentration of THG, dTHG, SJG, LMWH solution or blank control solution (Tris-BSA buffer Κ4) 30μ1 mixed with 1.0 IU/ml factor νΐΠ (30μ1), sequentially added kit reagent (30μ1), R ₂ (30μ1), incubate for 1 min at 37 °C, add R ₃ (30μ1), incubate for 1 min at 37 °C, stop the reaction with 20% acetic acid (60μ1) and detect OD _405nm . The AOD was calculated from the blank control (R4), and the IC ₅₀ value of each sample was determined to inhibit f.Xase according to the formula provided in the literature (Sheehan JP & Walke Ε. Κ,, Blood, 2006, 107: 3876-3882).

(2) Detection of HC-II-dependent antithrombin activity: Serial concentration of THG, dTHG, SJG, LMWH solution or blank control solution (Tris buffer) 50μ1 was added to 96-well microtiter plate, and 50μ1 was added. Ι μιηοΐ/L HC-Π, mixed, 37. Incubate for 2 min, then add 50 μl 5 U/ml IIa, incubate at 37 °C for 2 min, add 50 μl 2 mmol/L CS-0138, mix, incubate for 1 min at 37 ° C, stop the reaction with 50% acetic acid (ΙΟΟμΙ) and detect OD _{4 () 5 nm} . The AOD was calculated from the blank control (R4), and the IC ₅ value of each sample was determined to suppress Ila according to the formula provided in the literature (Sheehan JP & Walke Ε. Κ·, Blood, 2006, 107: 3876-3882).

 (3) Activity detection for prolonging APTT time: serial concentration of THG, dTHG, SJG, LMWH solution or blank control solution (Tris buffer) ΙΟμΙ mixed into 180μ1 rabbit plasma, and then measuring the time of each sample according to the kit instructions According to the test results of each sample, the drug concentration of the doubling time (doubling the time of sputum) was calculated.

 Results: See Table 5 and Figure 5.

 Table 5. Anticoagulant activity of THG/dTHG and reference samples

IC ₅₀ ( g/ml) multiplying APTT against f.Xase-extension

 anti-f.Xase-resistant

 Sample molecular weight

 Drug concentration APTT potency ratio

Mw (Da) Ila titer ratio [ ^li

 anti-f.Xase anti-Ila ^g/ml) [21

 THG 65820 0.275 1.25 4.54 1.77 8.66 dTHG-1 46070 0.270 1.56 5.78 2.05 7.59 dTHG-2 33260 0.268 1.64 6.1 1 2.50 9.32 dTHG-3 25380 0.266 1.75 6.58 3.28 12.3 dTHG-4 19650 0.253 1.90 7.51 3.59 14.2 dTHG-5 17150 0.245 2.27 9.26 4.08 16.6 dTHG-6 13950 0.216 2.84 13.1 4.56 21.1 dTHG-7 1 1580 0.282 3.55 12.6 6.29 22.3 dTHG-8 10260 0.463 4.02 8.68 8.82 19.1 19.1 dTHG-9 8549 0.714 5.96 8.34 10.6 14.8 dTHG-10 6725 0.864 6.23 2.17 21.9 7.6

 SJG-2 9300 0.683 5.35 7.83 9.7 14.2

 LMWH 3500-5500 9.22 6.08 0.66 4.48 0.48

^[1] Anti-f.Xase-anti-Ila potency ratio: anti-Ila IC ₅₀ (μ^ιηΐ) I anti-f.Xase IC ₅₀ (μ^ηιΐ)

[ ^2] Anti-f.Xase-prolonged APTT potency ratio: drug concentration of multiplied APTT^g/ml)/anti-f.Xase IC ₅₀ (g/ml) Literature (GZ. Feuerstein, et al., Arterioscler Thromb Vase Biol., 1999,

19:2554; CJ. Refino, et al., Arterioscler Thromb Vase Biol., 2002, 22: 517) shows that f. IXa inhibitors can significantly inhibit thrombosis at doses that do not substantially affect blood APTT and bleeding time. And the bleeding tendency of anticoagulant drugs is related to its antithrombin activity. In addition, the clinical practice of low molecular weight heparin has confirmed that as the anti-Xa/anti-Ila potency ratio increases, the bleeding tendency is significantly reduced (G. Andriuoli et al. Heamostasis, 1985, 15: 324). Based on the above understanding and the correlation between the molecular weight of dTHG and its potency at different targets of coagulation factors, appropriate depolymerization THG may produce the highest possible potency ratio of anti-f.Xase to HC-II-dependent anti-Ila activity and/or as high as possible anti-f.Xase and prolongation while eliminating FGAG-induced platelet aggregation activity. The potency ratio of APTT activity.

 The results in Table 5 show that THG and/or dTHG have prolonged APTT time, inhibit endogenous f.Xase and HC-II-dependent antithrombin activity, and depolymerization of THG can eliminate platelet-inducing activity, but the degree of depolymerization is It has different effects on anti-f.Xase, anti-Ila (dependent on HC-II) and its prolongation of APTT activity.

 First, the platelet-inducing activity of dTHG has a molecular weight (Mw) as low as about 20000 Da, and the anticoagulant activity (prolonged APTT time) is still present when the Mw is as low as about 6000 Da. Obviously, dTHG in this molecular weight range is more likely to have better application value than prototype THG.

Second, both THG and dTHG have potent inhibitory activity against endogenous f.Xase. Under the system conditions described in Example 3.2 of the present invention, for dTHG (or THG) having a weight average molecular weight of about 8,000 - 70,000 Da, it inhibits IC ₅ of f.Xase. The value is generally less than about 0.1 mol/L (less than about 1 g/ml) and generally shows a tendency for activity to increase with increasing molecular weight, but this trend is quite different in different molecular weight ranges. When the molecular weight is less than about 12000 Da, the activity of dTHG inhibiting f.Xase can be significantly reduced with the decrease of molecular weight; and when the molecular weight is not lower than about 12000 Da, the IC ₅ Q value is about 0.2 g/ml ~ 0.3 g. In the range of /ml, the IC ₅₀ value may decrease slightly by 4 according to the molecular weight in terms of molar concentration; but the IC ₅ c value of the mass concentration does not change substantially with the molecular weight, or more accurately It is said that it increases slightly with the increase in molecular weight. In general, for dTHG (or THG) f.Xase, when the molecular weight is not lower than about 10,000 Da, its activity of inhibiting endogenous factor X enzyme can be kept relatively constant to some extent.

The results in Table 5 also show that THG/dTHG has HC-II antithrombin activity and its activity to prolong APTT time. For dTHG (or THG) with a weight average molecular weight of about 8000 ~ 70,000 Da, its dependence on HC-II antithrombin activity and its prolonged APTT activity are enhanced with increasing molecular weight, which is manifested in its dependence on HC-II resistance. Thrombin activity of IC ₅ . Both the value and the drug concentration at which it multiplied the APTT time decreased linearly as the logarithm of the molecular weight increased.

 Based on the above findings of the present invention, it can be judged that when the weight average molecular weight of dTHG reaches about 10,000 Da, especially about 12,000 Da or more, the molecular weight affects the dTHG-prolonging APTT and the HCII-dependent antithrombin activity much more than the inhibition. The effect of the source of f.Xase activity. For convenience of description, Table 5 defines "anti-f.Xase-anti-Ila potency ratio" and "anti-f.Xase-prolonged APTT potency ratio" to reflect the anticoagulant activity of different molecular weight THG/dTHG. In general, for dTHG having a weight average molecular weight of not less than 10,000 Da, the lower the molecular weight, the higher the potency ratio of f.Xase activity to its dependence on HC-II antithrombin activity or prolonging APTT time activity. When the molecular weight is lower (less than about 10,000 Da), the potency ratio can show a trend of falling.

The results in Table 5 also indicate that LMWH has a relatively weak anti-f.Xase activity, whereas for dSJG, platelet-inducing activity limits the use of products with higher activity and/or higher "potency ratio". The present invention is based on (1) eliminating platelet-inducing activity, (2) obtaining as high an anti-Xase/anti-Ila potency ratio as possible, and/or (3) obtaining as high an anti-Xase/prolonged APTT potency ratio as possible, To comprehensively measure the relationship between the molecular weight of dTHG and its inhibition of f.Xase activity, anti-thrombin activity dependent on HC-II, prolongation of APTT time activity, and platelet-influencing activity, the weight average molecular weight (Mw) of dTHG selected in the present invention may range from about 8000 °. The preferred molecular weight range is from about 10,000 to 18,000 Da, and the more preferred molecular weight range is from about 12,000 to 16,000 Da.

3.3 dTHG anti-in vivo vein thrombosis

 Sample: dTHG-6, source see Example 1; low molecular weight heparin sodium (LMWH): 3500 ~ 5500 Da, 0.4 mix 4000 AxalU, Anofi - Aventis

 Method:

 (1) Rabbit venous thrombosis: Male New Zealand white rabbits were anesthetized with pentobarbital (30 mg/kg iv), and the left and right jugular veins were separated. Two ligation sutures were placed in the 2 cm section, and recombinant human tissue factor was injected intravenously. Lng/kg, 5 min after ligation of the proximal and distal end of the vein, 15 min after longitudinal incision of the blood vessel to remove the thrombus, filter paper washed dry blood, weighing the wet weight of the thrombus. The test drugs (dTHG-6, LMWH) and the control solvent (normal saline, NS) were administered subcutaneously 2 hours before the venous ligation.

 (2) Bleeding time test: SD rats, pentobarbital (30mg/kg) anesthesia, intravenous dTHG-6 or LMWH 15 sec after tailing (5mm from the end), carefully sucked off with filter paper every 15 sec Bleeding blot at the tail. It is judged that hemostasis has been stopped when there is no bleeding blot for 1 min.

 Result:

 (1) Rabbit venous thrombosis: In the model of venous thrombosis under ligation and hyperviscosity, subcutaneous injection of dTHG-64.5, 9, 18 mg/kg can significantly inhibit thrombosis, and the inhibition rate ranges from about 35% to about 70%. Its inhibitory activity showed a significant dose-effect relationship (Table 6). In this trial, LMWH 720 IU/kg inhibited venous thrombosis by approximately 56°/. .

 Table 6. Effect of THG on jugular vein thrombosis

 Fine n Thrombosis weight (mg) inhibition rate (%) Model group 10 152.8 ± 13.6 --

1HG 4.5 mgkg 10 98.3 ±25.6* 35.7

 9 mgkg 10 56.4 ± 20.6 * 63.1

 18 mgkg 10 45.7 ± 26.8 * 70.1

 LMWH 720 IUkg 10 67.3 ±21.2* 56.0

 Compared with the model group: * <0.05, ** <0.01

 (2) Detection of bleeding time: The effect of subcutaneous injection of dTHG-6 or LMWH on the bleeding time of rats. The results of the trial showed that dTHG-6 had a lower effect on bleeding time than the LMWH at the approximate anticoagulant and antithrombotic doses.

Claims

Claim

 1. An oligomeric pineapple glycosaminoglycan and a pharmaceutically acceptable salt thereof, wherein the oligomeric pineapple glycosaminoglycan is derived from fucosylated glycosaminoglycans of Thelenota ananas a depolymerization product of sugar having a weight average molecular weight of 8000 to 20000 Da, and a monosaccharide composition thereof including N-acetylgalactosamine, glucuronic acid, fucose, and a sulfate thereof, in terms of a molar ratio, N-acetylamino group Galactose: Glucuronic acid: Fucose: The ratio of sulfate groups represented by -OSO is '1: (1±0.3): (1±0.3): (3.5±0.5).

 The oligo-6-glycosaminoglycan and the pharmaceutically acceptable salt thereof according to claim 1, wherein the fucose sulfate group comprises 3-sulfate-rock in the monosaccharide composition Alginose base.

 The oligomeric pineapple glycosaminoglycan and the pharmaceutically acceptable salt thereof according to claim 1, wherein the oligomeric pineapple glycosaminoglycan has a weight average molecular weight of 10,000 to 18,000 Da.

 A method for producing the oligomeric pineapple glycosaminoglycan according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the preparation of the oligomeric pineapple glycosaminoglycan comprises the steps of:

 1) obtaining fucosylated glycosaminoglycan from the pineapple wall;

 2) utilizing the fucosylated glycosaminoglycan obtained by the peroxide depolymerization step 1) to obtain an oligomeric fucosylated glycosaminoglycan;

 3) Collect and purify oligomeric fucosylated glycosaminoglycans of the desired molecular weight range.

 The method according to claim 4, wherein in the presence of a catalyst, the peroxide depolymerizes the pineapple ginseng fucosylated glycosaminoglycan in the aqueous phase shield to obtain the oligomeric fucosylated glycosaminoglycan. Sugar, the catalyst is a catalyst containing a transition metal ion selected from the fourth period of the periodic table.

6. The method as claimed in claim 5, wherein said fourth period of the periodic table containing selected transition metal ion catalyst is ^{Cu +, Cu 2+, Fe 2+} , Fe 3+, Cr 3+, Cr 2 0 ₇ ² \Inorganic or organic salts formed by Mn ²⁺ , Zn ²⁺ , Ni ²⁺ , or a combination thereof.

 A pharmaceutical composition comprising the oligomeric pine ginseng glycosaminoglycan according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

 The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable salt is a sodium salt, a potassium salt or a calcium salt.

 The pharmaceutical composition according to claim 7 or 8, wherein the pharmaceutical composition is prepared as a lyophilized powder for injection.

 10. Use of a pharmaceutical composition according to claim 7 or 8 for the preparation of a medicament for the prevention and/or treatment of antithrombotic drugs.